Dissociated Dorsal Root Ganglion Research Articles

Histamine H4 receptor and TRPV1 mediate itch induced by cadaverine, a metabolite of the microbiome.

Cadaverine is an endogenous metabolite produced by the gut microbiome with various activity in physiological and pathological conditions. However, whether cadaverine regulates pain or itch remains unclear. In this study, we first found that cadaverine may bind to histamine 4 receptor (H4R) with higher docking energy score using molecular docking simulations, suggesting cadaverine may act as an endogenous ligand for H4R. We subsequently found intradermal injection of cadaverine into the nape or cheek of mice induces a dose-dependent scratching response in mice, which was suppressed by a selective H4R antagonist JNJ-7777120, transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine and PLC inhibitor U73122, but not H1R antagonist or TRPA1 antagonist or TRPV4 antagonist. Consistently, cadaverine-induced itch was abolished in Trpv1-/- but not Trpa1-/- mice. Pharmacological analysis indicated that mast cells and opioid receptors were also involved in cadaverine-induced itch in mice. scRNA-Seq data analysis showed that H4R and TRPV1 are mainly co-expressed on NP2, NP3 and PEP1 DRG neurons. Calcium imaging analysis showed that cadaverine perfusion enhanced calcium influx in the dissociated dorsal root ganglion (DRG) neurons, which was suppressed by JNJ-7777120 and capsazepine, as well as in the DRG neurons from Trpv1-/- mice. Patch-clamp recordings found that cadaverine perfusion significantly increased the excitability of small diameter DRG neurons, and JNJ-7777120 abolished this effect, indicating involvement of H4R. Together, these results provide evidences that cadaverine is a novel endogenous pruritogens, which activates H4R/TRPV1 signaling pathways in the primary sensory neurons.

Repeated closed-head mild traumatic brain injury-induced inflammation is associated with nociceptive sensitization.

Individuals who have experienced mild traumatic brain injuries (mTBIs) suffer from several comorbidities, including chronic pain. Despite extensive studies investigating the underlying mechanisms of mTBI-associated chronic pain, the role of inflammation in long-term pain after mTBIs is not fully elucidated. Given the shifting dynamics of inflammation, it is important to understand the spatial-longitudinal changes in inflammatory processes following mTBIs and their effects on TBI-related pain. We utilized a recently developed transgenic caspase-1 luciferase reporter mouse model to monitor caspase-1 activation through a thinned skull window in the in vivo setting following three closed-head mTBI events. Organotypic coronal brain slice cultures and acutely dissociated dorsal root ganglion (DRG) cells provided tissue-relevant context of inflammation signal. Mechanical allodynia was assessed by mechanical withdrawal threshold to von Frey and thermal hyperalgesia withdrawal latency to radiant heat. Mouse grimace scale (MGS) was used to detect spontaneous or non-evoked pain. In some experiments, mice were prophylactically treated with MCC950, a potent small molecule inhibitor of NLRP3 inflammasome assembly to inhibit injury-induced inflammatory signaling. Bioluminescence spatiotemporal dynamics were quantified in the head and hind paws, and caspase-1 activation was confirmed by immunoblot. Immunofluorescence staining was used to monitor the progression of astrogliosis and microglial activation in ex vivo brain tissue following repetitive closed-head mTBIs. Mice with repetitive closed-head mTBIs exhibited significant increases of the bioluminescence signals within the brain and paws in vivo for at least one week after each injury. Consistently, immunoblotting and immunofluorescence experiments confirmed that mTBIs led to caspase-1 activation, astrogliosis, and microgliosis. Persistent changes in MGS and hind paw withdrawal thresholds, indicative of pain states, were observed post-injury in the same mTBI animals in vivo. We also observed enhanced inflammatory responses in ex vivo brain slice preparations and DRG for at least 3days following mTBIs. In vivo treatment with MCC950 significantly reduced caspase-1 activation-associated bioluminescent signals in vivo and decreased stimulus-evoked and non-stimulus evoked nociception. Our findings suggest that the inflammatory states in the brain and peripheral nervous system following repeated mTBIs are coincidental with the development of nociceptive sensitization, and that these events can be significantly reduced by inhibition of NLRP3 inflammasome activation.

MP27-01 TARGETED DEPLETION OF FIDGETIN-LIKE 2 (FL2) PROMOTES NERVE REGENERATION AND IMPROVES BOTH ERECTILE AND BLADDER FUNCTION OUTCOMES IN A RODENT MODEL OF RADICAL PROSTATECTOMY

You have accessJournal of UrologyCME1 Apr 2023MP27-01 TARGETED DEPLETION OF FIDGETIN-LIKE 2 (FL2) PROMOTES NERVE REGENERATION AND IMPROVES BOTH ERECTILE AND BLADDER FUNCTION OUTCOMES IN A RODENT MODEL OF RADICAL PROSTATECTOMY Lisa Baker, Moses Tar, Yi Wang, David Sharp, Sylvia Suadicani, and Kelvin Davies Lisa BakerLisa Baker More articles by this author , Moses TarMoses Tar More articles by this author , Yi WangYi Wang More articles by this author , David SharpDavid Sharp More articles by this author , Sylvia SuadicaniSylvia Suadicani More articles by this author , and Kelvin DaviesKelvin Davies More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Damage to the cavernous nerves (CN) during radical prostatectomy (RP) commonly results in urogenital dysfunction, which includes bladder and erectile dysfunction (ED). We recently identified fidgetin-like 2 (FL2) as a negative regulator of microtubule dynamics which when depleted in nerve cells in vitro accelerates axonal outgrowth, and in an in vivo model of RP promotes nerve regeneration and improves erectile function outcomes. We have expanded upon these studies to determine if FL2 depletion might also improve bladder function outcomes following CN injury. METHODS: To study the effects of FL2 depletion on axon growth and on the microtubule (MT) cytoskeleton within the axon shaft in vitro, dissociated dorsal root ganglion (DRG) neurons were transduced with AAV5 containing a plasmid encoding FL2 or scrambled small hairpin RNA (shRNA) and a GFP reporter. Five-days after treatment, neurons were assessed for neurite growth, fixed and immunostained for MT. As a model of CN injury, rats underwent CN transection. At the site and time of injury, control- or FL2-siRNA was applied. Voiding function was evaluated 1 week post-injury, using the voided stain on paper (VSOP) method. At 2 weeks CN regeneration was assessed visually and erectile function was assessed by cavernosometry. RESULTS: In vitro FL2 knockdown in neurons enhanced average neurite length and growth rate. Animals treated withFL2-siRNA had significantly lower voiding frequency and higher voided volume per micturition when compared to animals treated with control-siRNA. There was visible regeneration of the CN in 7 out of 8 animals treated with FL2-siRNA (with no observed regeneration in animals treated with control-siRNA). In animals in which there was sufficient CN regeneration to allow electrostimulation, FL2-siRNA treated animals had significantly improved erectile function compared to controls. CONCLUSIONS: FL2-depletion after CN injury results in visible regeneration of the CN, with improved erectile and bladder function outcomes. Our work suggests that depleting FL2 promotes nerve regeneration through directly enhancing the growth rate of injured axons. Overall, we have identified FL2 as a promising therapeutic target for promoting CN regeneration and functional recovery of urogenital function after iatrogenic nerve injury resulting from the surgical procedures of RP. Source of Funding: NIH/NIDDK © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e362 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lisa Baker More articles by this author Moses Tar More articles by this author Yi Wang More articles by this author David Sharp More articles by this author Sylvia Suadicani More articles by this author Kelvin Davies More articles by this author Expand All Advertisement PDF downloadLoading ...

Acid-sensing ion channel 3 is required for agmatine-induced histamine-independent itch in mice.

Itch is a common symptom of many skin and systemic diseases. Identifying novel endogenous itch mediators and the downstream signaling pathways involved will contribute to the development of new strategies for the treatment of chronic itch. In the present study, we adopted behavioral testing, patch clamp recording and metabonomics analysis to investigate the role of agmatine in itch and the underlying mechanism. Behavioral analysis was used to evaluate the establishing of acute and chronic itch mice model, and to test the effects of different drugs or agents on mice itch behavior. Western blotting analysis was used to test the effect of agmatine on phosphorylation of ERK (p-ERK) expression in the spinal cord. Patch clamp recording was used to determine the effect agmatine on the excitability of DRG neurons and the role of ASIC3. Finally, the metabonomics analysis was performed to detect the concentration of agmatine in the affected skin under atopic dermatitis or psoriasis conditions. We fused a mouse model and found that an intradermal injection of agmatine (an endogenous polyamine) into the nape of the neck or cheek induced histamine-independent scratching behavior in a dose-dependent manner. In addition, the ablation of nociceptive C-fibers by resiniferatoxin (RTX) abolished agmatine-induced scratching behavior. However, agmatine-induced itch was not affected by the pharmacological inhibition of either transient receptor potential vanilloid 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1); similar results were obtained from TRPV1-/- or TRPA1-/- mice. Furthermore, agmatine-induced itch was significantly suppressed by the administration of acid-sensing ion channel 3 (ASIC3) inhibitors, APETx2 or amiloride. Agmatine also induced the upregulation of p-ERK in the spinal cord; this effect was inhibited by amiloride. Current clamp recording showed that the acute perfusion of agmatine reduced the rheobase and increased the number of evoked action potentials in acute dissociated dorsal root ganglion (DRG) neurons while amiloride reversed agmatine-induced neuronal hyperexcitability. Finally, we identified significantly higher levels of agmatine in the affected skin of a mouse model of atopic dermatitis (AD) when compared to controls, and the scratching behavior of AD mice was significantly attenuated by blocking ASIC3. Collectively, these results provide evidence that agmatine is a novel mediator of itch and induces itch via the activation of ASIC3. Targeting neuronal ASIC3 signaling may represent a novel strategy for the treatment of itch.

Effect of high-frequency membrane potential alternation between depolarization and hyperpolarization on dorsal root ganglion neurons of rats.

The purpose of this study was to determine how sensory neurons respond to high-frequency membrane potential alternation between depolarization and hyperpolarization. Membrane currents were recorded from dissociated dorsal root ganglion (DRG) neurons of adult rats using the whole cell patch clamp technique in voltage clamp mode. Stepwise depolarization of the membrane was applied first to determine the threshold membrane potential for inducing an action potential (AP) current. Then, membrane potential alternation between depolarization (to +20 mV) and hyperpolarization (to -110 mV) was applied to the neuron for 10s at different frequencies (10 Hz to 1 kHz). The tested DRG neurons had APs of either a long duration (>10ms) or a short duration (<10ms). Membrane potential alternation at ≥500 Hz completely disrupted the AP generation, disabled the ion channel gating function, and produced membrane current alternating symmetrically across zero. Replacing extracellular sodium with potassium increased the amplitude of the membrane current response and caused the membrane current to be larger during hyperpolarization than during depolarization. These results support the hypothesis that high-frequency biphasic stimulation blocks axonal conduction by driving the potassium channel open constantly. Understanding neural membrane response to high-frequency membrane potential alternation is important to reveal the possible mechanisms underlying axonal conduction block induced by high-frequency biphasic stimulation.

Real Time Imaging of Mitochondrial Dynamics in the Dorsal Root Ganglion Following Nerve Injury

Mitochondria are dynamic and motile organelles that respond rapidly to oxidative stress and cellular energy demands. Their morphological changes, as determined by fission and fusion, have been linked to the progression of neuropathic pain. However, little is known about specific pathology underlying mitochondrial dysfunction after peripheral nerve injury or how pharmacological interventions affect these changes. Our goal is to evaluate mitochondrial dynamics following peripheral nerve injury by characterizing axonal mitochondrial density, movement in axons and fusion-fission events. We dissociated dorsal root ganglia (DRG) from mice with spare nerve injury (SNI) and selectively labelled mitochondria with a live-cell fluorescent dye. Time lapse videos were then captured using a spinning disk confocal microscope. We observed a significant increase in axonal mitochondrial density in DRG cultures obtained from SNI mice when compared to naïve cultures. Representative kymograph images depict a larger proportion of mitochondria moving anterogradely in axons of DRG neurons from SNI mice. Interestingly, we found that the SNI-mitochondria are trafficked at lower velocities. Previous studies have shown that calcium overload in damaged mitochondria may contribute to decreased mitochondrial motility. Additionally, we observe a decrease in perinuclear clustering within the SNI group which may indicate a disruption in mitochondrial networking or a failure to regulate fission events in DRG neurons after injury. Collectively, our findings highlight the benefit of real time imaging of DRG neurons to gain greater insight into specific aspects of mitochondrial dysfunction after nerve injury. Our findings likely have important implications for therapeutic approaches to neuropathic pain. Supported by NIH grant NS065926.

Allium macrostemon Bunge. exerts analgesic activity by inhibiting NaV1.7 channel

Ethnopharmacological relevanceAllium macrostemon Bunge. is an edible Chinese herb traditionally used for the treatment of thoracic pain, stenocardia, heart asthma and diarrhea. Although its biological potential has been extensively proven such as antioxidant activity, antiplatelet aggregation, vasodilation and antidepressant-like activity, there are no reports in the literature regarding its pharmacological analgesic activity. Aim of the studyThe study was carried out to examine the anti-nociceptive activity of the crude extract of A. macrostemon bulbs and interpret its likely molecular target. Materials and methodsThe bulbs of A. macrostemon were gathered, dried-up, and extracted with water (AMWD). AMWD was subjected to activity testing, using chemical-induced (acetic acid and formalin test) and heat-induced (hot plate) pain models. To evaluate the likely mechanistic strategy involved in the analgesic effect of AMWD, whole-cell patch clamp recordings were conducted in acutely dissociated dorsal root ganglion (DRG) neurons and human embryonic kidney 293T (HEK293T) cells expressing pain-related receptors. Electrophysiological methods were employed to detect the action potentials of DRG neurons and potential targets of A. macrostemon. ResultsAMWD showed significant palliative effect in all heat and chemical induced pain assays. Moreover, AMWD significantly reduces the excitability of dorsal root ganglion neurons by reducing the firing frequency of action potentials. Further analysis revealed that voltage-gated sodium channel Nav1.7 is the potential target of A. macrostemon for its analgesic activity. ConclusionThis study has brought new scientific evidence of preclinical efficacy of A. macrostemon as an anti-nociceptive agent. Apparently, these effects are involved with the inhibition of the voltage-sensitive Nav1.7 channel contributing to the reduction of peripheral neuronal excitability. Our present study justifies the folkloric usage of A. macrostemon as a remedy for several pain states. Furthermore, A. macrostemon is a good resource for the development of analgesic drugs targeting Nav1.7 channel.

Retracing Schwann Cell Developmental Transitions in Embryonic Dissociated DRG/Schwann Cell Cocultures in Mice.

Embryonic Dissociated Dorsal Root Ganglia (DRG) cultures are often used to investigate the role of novel molecular pathways or drugs in Schwann cell development and myelination. These cultures largely recapitulate the order of cellular and molecular events that occur in Schwann cells of embryonic nerves. However, the timing of Schwann cell developmental transitions, notably the transition from Schwann Cell Precursors (SCP) to immature Schwann cells (iSC) and then to myelinating Schwann cells, has not been estimated so far in this culture system. In this study, we determined the expression profiles of Schwann cell developmental genes during the first week of culture and then compared our data to the expression profiles of these genes in developing spinal nerves. This helped in identifying that SCP transition into iSC between the 5th and 7th day in vitro. Furthermore, we also investigated the transition of immature cells into pro-myelinating and myelinating Schwann cells upon the induction of myelination in vitro. Our results suggest that Schwann cell differentiation beyond the immature stage can be observed as early as 4 days post the induction of myelination in cocultures. Finally, we compared the myelinating potential of coculture-derived Schwann cell monocultures to cultures established from neonatal sciatic nerves and found that both these culture systems exhibit similar myelinating phenotypes. In effect, our results allow for a better understanding and interpretation of coculture experiments especially in studies that aim to elucidate the role of a novel actor in Schwann cell development and myelination.

Aligned Fingolimod-Releasing Electrospun Fibers Increase Dorsal Root Ganglia Neurite Extension and Decrease Schwann Cell Expression of Promyelinating Factors.

Researchers are investigating the use of biomaterials with aligned guidance cues, like those provided by aligned electrospun fibers, to facilitate axonal growth across critical-length peripheral nerve defects. To enhance the regenerative outcomes further, these aligned fibers can be designed to provide local, sustained release of therapeutics. The drug fingolimod improved peripheral nerve regeneration in preclinical rodent models by stimulating a pro-regenerative Schwann cell phenotype and axonal growth. However, the systemic delivery of fingolimod for nerve repair can lead to adverse effects, so it is necessary to develop a means of providing sustained delivery of fingolimod local to the injury. Here we created aligned fingolimod-releasing electrospun fibers that provide directional guidance cues in combination with the local, sustained release of fingolimod to enhance neurite outgrowth and stimulate a pro-regenerative Schwann cell phenotype. Electrospun fiber scaffolds were created by blending fingolimod into poly(lactic-co-glycolic acid) (PLGA) at a w/w% (drug/polymer) of 0.0004, 0.02, or 0.04%. We examined the effectiveness of these scaffolds to stimulate neurite extension in vitro by measuring neurite outgrowth from whole and dissociated dorsal root ganglia (DRG). Subsequently, we characterized Schwann cell migration and gene expression in vitro. The results show that drug-loaded PLGA fibers released fingolimod for 28 days, which is the longest reported release of fingolimod from electrospun fibers. Furthermore, the 0.02% fingolimod-loaded fibers enhanced neurite outgrowth from whole and dissociated DRG neurons, increased Schwann cell migration, and reduced the Schwann cell expression of promyelinating factors. The in vitro findings show the potential of the aligned fingolimod-releasing electrospun fibers to enhance peripheral nerve regeneration and serve as a basis for future in vivo studies.

A11 MICROBIAL AND METABOLOMIC PROFILES IDENTIFY SUBSETS OF DIARRHEA PREDOMINANT IRRITABLE BOWEL SYNDROME

Abstract Background Irritable bowel syndrome (IBS) patients are subtyped by predominant bowel habit rather than pathophysiological mechanisms and this may underlie challenges in identifying more effective targets for designing new treatments. Metabolomics and microbial analysis can distinguish IBS patients from healthy controls but it is unknown if they can identify pathophysiological IBS subgroups. Aims To phenotype subgroups of IBS patients using metabolomics and microbial analysis and determine if these subgroups have different underlying pain signalling mechanisms. Methods Symptom history, stool and urine were collected from 30 diarrhea predominant (IBS-D) and 30 constipation predominant (IBS-C) IBS patients (Rome IV). Liquid Chromatography-Mass Spectrometry quantified 130 metabolites in stool and urine. The GA-map™ Dysbiosis Test targeting ≥300 bacteria on different taxonomic levels was used to identify stool microbial composition. Multivariate OPLS discriminatory analysis assessed metabolomics and microbial profiles. To assess potential effects on pain signalling, the effect of stool supernatant on dissociated dorsal root ganglia (DRG) neuron responses to capsaicin (10nM) was assessed using Ca2+ imaging. Results Within both IBS-D and IBS-C, combined stool/urine metabolomic profiles of patients with a dysbiosis-like (DL) IBS (onset following antibiotics, enteric infection, or travel) were distinct from patients with a non-DL IBS onset (IBS-D R2=0.7, Q2=0.5; IBS-C R2=0.5, Q2=0.4); fecal glutamic acid and urinary pyruvic acid were the main metabolites driving separation. However, microbial profiles of DL vs non-DL onset could only be discriminated in IBS-D (R2=0.8, Q2=0.4). In the patients with a DL IBS onset, stool metabolomic profiles of the 7 IBS-C discriminated from the 8 IBS-D patients (R2=0.9, Q2=0.8). Profile differences were not seen between IBS-C and IBS-D with a non-DL onset of IBS. In preliminary studies, incubation of DRG neurons with stool supernatant from 1 DL IBS-D and 1 non-DL IBS-D increased peak [Ca2+]i responses to capsaicin compared to incubation with media (DL: 5.5±0.9 vs 2.3±0.7; non-DL: 6.9±0.7 vs 3.9±0.4% ΔF/F). Similarly, the number of responsive neurons to capsaicin was increased after incubation with IBS stool supernatant vs media (DL: 27% vs 8%; non-DL 19% vs 12%). Conclusions Different metabolomic and bacterial profiles between DL and non-DL onset of IBS-D suggests a novel means to better phenotype clinically defined IBS subgroups. While initial results with stool supernatants from both a DL and non-DL IBS-D patient suggest increased pain signalling in DRG neurons, more studies are needed to determine if there are differences between these two subgroups as well as healthy controls. Funding Agencies CIHRSoutheastern Ontario Academic Medical Organization (SEAMO)

A124 SYNERGISTIC INTERACTION OF CANNABINOIDS AND OPIOIDS REDUCES PAIN SIGNALING IN COLONIC NOCICEPTIVE NERVES

Abstract Background With the recent legalization of recreational marijuana in Canada, increasing numbers of patients with gastrointestinal (GI) disorders are using cannabis to treat their pain, either alone or together with opioids. However, little is known about potential benefits of cannabinoids for treating visceral pain originating within the GI tract and whether the combined use of cannabinoids and opioids could enable the reduction or even discontinuation of opioids. Aims To investigate the effects of cannabinoids alone or in combination with opioids on colonic nociceptive nerves. Methods Extracellular afferent nerve recordings were obtained from ex vivo flat sheet preparations of male C57BL/6 mouse distal colons. Single unit analysis discriminated individual afferent neuron responses to mechanical probing of the colon with a 1g von Frey hair before and after superfusion of HU-210, a selective CB1 receptor agonist, HU-308, a selective CB2 receptor agonist, DAMGO, a selective mu-opioid receptor (MOR) agonist, or a combination. In parallel studies, perforated patch clamp techniques were employed to assess the rheobase as a measure of neuronal excitability in acutely dissociated dorsal root ganglia (DRG) neurons in the presence of one or more of these agonists. Data were analyzed using a one-way ANOVA with Bonferroni multiple comparisons test. Results Superfusion of HU-210 (1 μM), caused significant inhibition in afferent nerve mechanosensitivity compared to control (6.2±1.1 vs. 13.7±2.5 Hz, p=0.005, n=10); lower concentrations (10 nM and 100 nM) had no effect (p&amp;gt;0.99, n=11; p=0.600, n=10 respectively). Conversely, the CB2 agonist HU-308 (1 μM and 10 μM), did not alter mechanosensitivity (p&amp;gt;0.9, n=8 for both concentrations). Superfusion of HU-210 alone (100 nM) or DAMGO (1 nM) alone in the same recording had no effect, but when both agonists were superfused together, there was a significant reduction in mechanosensitivity (8.1±1.7 vs. 14.8±2.3 Hz, p&amp;lt;0.01, n=10). In patch clamp recordings of DRG neurons, HU-210 (1 μM) decreased excitability (i.e. increased rheobase, 94.4±9.4 vs. 62.7±6.4 pA; p=0.031, n=9), whereas a lower concentration (100 nM) had no effect. Similar to afferent nerve recordings, when applied alone, DAMGO (1 nM) and HU-210 (100 nM) did not affect rheobase (DAMGO: p&amp;gt;0.99, n=9; HU-210: p&amp;gt;0.99, n=10), whereas the combination of both agonists significantly decreased excitability (123.0±13.4 vs. 62.7±6.4 pA, p&amp;lt;0.01, n=10). Conclusions Activation of CB1 receptors inhibits mechanosensitivity of colonic afferent nerves while a CB2 agonist had no effect. Interestingly, combination of sub-threshold concentrations of CB1 and MOR agonists inhibited colonic afferent nerves and thus, may suggest that cannabinoids could enable opioid dose reduction or discontinuation. Funding Agencies None

Collapsin Response Mediator Protein 4 (CRMP4) Facilitates Wallerian Degeneration and Axon Regeneration following Sciatic Nerve Injury.

In contrast to neurons in the CNS, damaged neurons from the peripheral nervous system (PNS) regenerate, but this process can be slow and imperfect. Successful regeneration is orchestrated by cytoskeletal reorganization at the tip of the proximal axon segment and cytoskeletal disassembly of the distal segment. Collapsin response mediator protein 4 (CRMP4) is a cytosolic phospho-protein that regulates the actin and microtubule cytoskeleton. During development, CRMP4 promotes growth cone formation and dendrite development. Paradoxically, in the adult CNS, CRMP4 impedes axon regeneration. Here, we investigated the involvement of CRMP4 in peripheral nerve injury in male and female Crmp4−/− mice following sciatic nerve injury. We find that sensory axon regeneration and Wallerian degeneration are impaired in Crmp4−/− mice following sciatic nerve injury. In vitro analysis of dissociated dorsal root ganglion (DRG) neurons from Crmp4−/− mice revealed that CRMP4 functions in the proximal axon segment to promote the regrowth of severed DRG neurons and in the distal axon segment where it facilitates Wallerian degeneration through calpain-dependent formation of harmful CRMP4 fragments. These findings reveal an interesting dual role for CRMP4 in proximal and distal axon segments of injured sensory neurons that coordinately facilitate PNS axon regeneration.

Resolvin D3 controls mouse and human TRPV1-positive neurons and preclinical progression of psoriasis.

Rationale: Psoriasis is a chronic inflammatory disease caused by a complex interplay between the immune and nervous systems with recurrent scaly skin plaques, thickened stratum corneum, infiltration and activation of inflammatory cells, and itch. Despite an increasing availability of immune therapies, they often have adverse effects, high costs, and dissociated effects on inflammation and itch. Activation of sensory neurons innervating the skin and TRPV1 (transient receptor potential vanilloid 1) are emerging as critical components in the pathogenesis of psoriasis, but little is known about their endogenous inhibitors. Recent studies have demonstrated that resolvins, endogenous lipid mediators derived from omega-3 fatty acids, are potent inhibitors of TRP channels and may offer new therapies for psoriasis without known adverse effects.Methods: We used behavioral, electrophysiological and biochemical approaches to investigate the therapeutic effects of resolvin D3 (RvD3), a novel family member of resolvins, in a preclinical model of psoriasis consisting of repeated topical applications of imiquimod (IMQ) to murine skin, which provokes inflammatory lesions that resemble human psoriasis.Results: We report that RvD3 specifically reduced TRPV1-dependent acute pain and itch in mice. Mechanistically, RvD3 inhibited capsaicin-induced TRPV1 currents in dissociated dorsal root ganglion (DRG) neurons via the N-formyl peptide receptor 2 (i.e. ALX/FPR2), a G-protein coupled receptor. Single systemic administration of RvD3 (2.8 mg/kg) reversed itch after IMQ, and repeated administration largely prevented the development of both psoriasiform itch and skin inflammation with concomitant decreased in calcitonin gene-related peptide (CGRP) expression in DRG neurons. Accordingly, specific knockdown of CGRP in DRG was sufficient to prevent both psoriasiform itch and skin inflammation similar to the effects following RvD3 administration. Finally, we elevated the translational potential of this study by showing that RvD3 significantly inhibited capsaicin-induced TRPV1 activity and CGRP release in human DRG neurons.Conclusions: Our findings demonstrate a novel role for RvD3 in regulating TRPV1/CGRP in mouse and human DRG neurons and identify RvD3 and its neuronal pathways as novel therapeutic targets to treat psoriasis.

TNF-\u03b1 mediated upregulation of NaV1.7 currents in rat dorsal root ganglion neurons is independent of CRMP2 SUMOylation

Clinical and preclinical studies have shown that patients with Diabetic Neuropathy Pain (DNP) present with increased tumor necrosis factor alpha (TNF-α) serum concentration, whereas studies with diabetic animals have shown that TNF-α induces an increase in NaV1.7 sodium channel expression. This is expected to result in sensitization of nociceptor neuron terminals, and therefore the development of DNP. For further study of this mechanism, dissociated dorsal root ganglion (DRG) neurons were exposed to TNF-α for 6 h, at a concentration equivalent to that measured in STZ-induced diabetic rats that developed hyperalgesia. Tetrodotoxin sensitive (TTXs), resistant (TTXr) and total sodium current was studied in these DRG neurons. Total sodium current was also studied in DRG neurons expressing the collapsin response mediator protein 2 (CRMP2) SUMO-incompetent mutant protein (CRMP2-K374A), which causes a significant reduction in NaV1.7 membrane cell expression levels. Our results show that TNF-α exposure increased the density of the total, TTXs and TTXr sodium current in DRG neurons. Furthermore, TNF-α shifted the steady state activation and inactivation curves of the total and TTXs sodium current. DRG neurons expressing the CRMP2-K374A mutant also exhibited total sodium current increases after exposure to TNF-α, indicating that these effects were independent of SUMOylation of CRMP2. In conclusion, TNF-α sensitizes DRG neurons via augmentation of whole cell sodium current. This may underlie the pronociceptive effects of TNF-α and suggests a molecular mechanism responsible for pain hypersensitivity in diabetic neuropathy patients.

Bisphenol A Regulates Sodium Ramp Currents in Mouse Dorsal Root Ganglion Neurons and Increases Nociception

17β-Estradiol mediates the sensitivity to pain and is involved in sex differences in nociception. The widespread environmental disrupting chemical bisphenol A (BPA) has estrogenic activity, but its implications in pain are mostly unknown. Here we show that treatment of male mice with BPA (50 µg/kg/day) during 8 days, decreases the latency to pain behavior in response to heat, suggesting increased pain sensitivity. We demonstrate that incubation of dissociated dorsal root ganglia (DRG) nociceptors with 1 nM BPA increases the frequency of action potential firing. SCN9A encodes the voltage-gated sodium channel Nav1.7, which is present in DRG nociceptors and is essential in pain signaling. Nav1.7 and other voltage-gated sodium channels in mouse DRG are considered threshold channels because they produce ramp currents, amplifying small depolarizations and enhancing electrical activity. BPA increased Nav-mediated ramp currents elicited with slow depolarizations. Experiments using pharmacological tools as well as DRG from ERβ−/− mice indicate that this BPA effect involves ERα and phosphoinositide 3-kinase. The mRNA expression and biophysical properties other than ramp currents of Nav channels, were unchanged by BPA. Our data suggest that BPA at environmentally relevant doses affects the ability to detect noxious stimuli and therefore should be considered when studying the etiology of pain conditions.

Knockdown siRNA Targeting the Mitochondrial Sodium-Calcium Exchanger-1 Inhibits the Protective Effects of Two Cannabinoids Against Acute Paclitaxel Toxicity.

Treatment with cannabidiol (CBD) or KLS-13019 (novel CBD analog), has previously been shown to prevent paclitaxel-induced mechanical allodynia in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). The mechanism of action for CBD- and KLS-13019-mediated protection now has been explored with dissociated dorsal root ganglion (DRG) cultures using small interfering RNA (siRNA) to the mitochondrial Na+ Ca2+ exchanger-1 (mNCX-1). Treatment with this siRNA produced a 50-55% decrease in the immunoreactive (IR) area for mNCX-1 in neuronal cell bodies and a 72-80% decrease in neuritic IR area as determined with high-content image analysis. After treatment with 100nM KLS-13019 and siRNA, DRG cultures exhibited a 75 ± 5% decrease in protection from paclitaxel-induced toxicity; whereas siRNA studies with 10μM CBD produced a 74 ± 3% decrease in protection. Treatment with mNCX-1 siRNA alone did not produce toxicity. The protective action of cannabidiol and KLS-13019 against paclitaxel-induced toxicity during a 5-h test period was significantly attenuated after a 4-day knockdown of mNCX-1 that was not attributable to toxicity. These data indicate that decreases in neuritic mNCX-1 corresponded closely with decreased protection after siRNA treatment. Pharmacological blockade of mNCX-1 with CGP-37157 produced complete inhibition of cannabinoid-mediated protection from paclitaxel in DRG cultures, supporting the observed siRNA effects on mechanism.

Structure-Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.

α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABAB receptors (GABABR). Therefore, it is unclear whether activation of GABABR or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABABR over α9α10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABABR-mediated inhibition of high voltage-activated (HVA) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Difference of acute dissociation and 1-day culture on the electrophysiological properties of rat dorsal root ganglion neurons.

The dissociated dorsal root ganglion (DRG) neurons with or without culture were widely used for investigation of their electrophysiological properties. The culture procedures, however, may alter the properties of these neurons and the effects are not clear. In the present study, we recorded the action potentials (AP) and the voltage-gated Na+, K+, and Ca2+ currents with patch clamp technique and measured the mRNA of Nav1.6-1.9 and Cav2.1-2.2 with real-time PCR technique from acutely dissociated and 1-day (1-d) cultured DRG neurons. The effects of the nerve growth factor (NGF) on the expression of Nav1.6-1.9 and Cav2.1-2.2 were evaluated. The neurons were classified as small (DRG-S), medium (DRG-M), and large (DRG-L), according to their size frequency distribution pattern. We found 1-d culture increased the AP size but reduced the excitability, and reduced the voltage-gated Na+ and Ca2+ currents and their corresponding mRNA expression in all types of neurons. The lack of NGF in the culture medium may contribute to the reduced Na+ and Ca2+ current, as the application of NGF recovered some of the reduced transcripts (Nav1.9, Cav2.1, and Cav2.2). 1-d culture showed neuron-type specific effects on some of the AP properties: it increased the maximum AP depolarizing rate (MDR) and hyperpolarized the resting membrane potential (RP) in DRG-M and DRG-L neurons, but slowed the maximum AP repolarizing rate (MRR) in DRG-S neurons. In conclusion, the 1-d cultured neurons had different properties with those of the acutely dissociated neurons, and lack of NGF may contribute to some of these differences.

Prolonged controlled delivery of nerve growth factor using porous silicon nanostructures

Although nerve growth factor (NGF) is beneficial for the treatment of numerous neurological and non-neurological diseases, its therapeutic administration represents a significant challenge, due to the difficulty to locally deliver relevant doses in a safe and non-invasive manner. In this work, we employ degradable nanostructured porous silicon (PSi) films as carriers for NGF, allowing its continuous and prolonged release, while retaining its bioactivity. The PSi carriers exhibit high loading efficacy (up to 90%) of NGF and a continuous release, with no burst, over a period of>26days. The released NGF bioactivity is compared to that of free NGF in both PC12 cells and dissociated dorsal root ganglion (DRG) neurons. We show that the NGF has retained its bioactivity and induces neurite outgrowth and profound differentiation (of >50% for PC12 cells) throughout the period of release within a single administration. Thus, this proof-of-concept study demonstrates the immense therapeutic potential of these tunable carriers as long-term implants of NGF reservoirs and paves the way for new localized treatment strategies of neurodegenerative diseases.

Combined Changes in Chloride Regulation and Neuronal Excitability Enable Primary Afferent Depolarization to Elicit Spiking without Compromising its Inhibitory Effects.

The central terminals of primary afferent fibers experience depolarization upon activation of GABAA receptors (GABAAR) because their intracellular chloride concentration is maintained above electrochemical equilibrium. Primary afferent depolarization (PAD) normally mediates inhibition via sodium channel inactivation and shunting but can evoke spikes under certain conditions. Antidromic (centrifugal) conduction of these spikes may contribute to neurogenic inflammation while orthodromic (centripetal) conduction could contribute to pain in the case of nociceptive fibers. PAD-induced spiking is assumed to override presynaptic inhibition. Using computer simulations and dynamic clamp experiments, we sought to identify which biophysical changes are required to enable PAD-induced spiking and whether those changes necessarily compromise PAD-mediated inhibition. According to computational modeling, a depolarizing shift in GABA reversal potential (EGABA) and increased intrinsic excitability (manifest as altered spike initiation properties) were necessary for PAD-induced spiking, whereas increased GABAAR conductance density (ḡGABA) had mixed effects. We tested our predictions experimentally by using dynamic clamp to insert virtual GABAAR conductances with different EGABA and kinetics into acutely dissociated dorsal root ganglion (DRG) neuron somata. Comparable experiments in central axon terminals are prohibitively difficult but the biophysical requirements for PAD-induced spiking are arguably similar in soma and axon. Neurons from naïve (i.e. uninjured) rats were compared before and after pharmacological manipulation of intrinsic excitability, and against neurons from nerve-injured rats. Experimental data confirmed that, in most neurons, both predicted changes were necessary to yield PAD-induced spiking. Importantly, such changes did not prevent PAD from inhibiting other spiking or from blocking spike propagation. In fact, since the high value of ḡGABA required for PAD-induced spiking still mediates strong inhibition, we conclude that PAD-induced spiking does not represent failure of presynaptic inhibition. Instead, diminished PAD caused by reduction of ḡGABA poses a greater risk to presynaptic inhibition and the sensory processing that relies upon it.